CN109694400A - A kind of expression respiratory syncystial virus F protein and preparation method thereof - Google Patents

A kind of expression respiratory syncystial virus F protein and preparation method thereof Download PDF

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Publication number
CN109694400A
CN109694400A CN201910095604.1A CN201910095604A CN109694400A CN 109694400 A CN109694400 A CN 109694400A CN 201910095604 A CN201910095604 A CN 201910095604A CN 109694400 A CN109694400 A CN 109694400A
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protein
cell
leu
ser
lys
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高光
崔院平
程远政
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Suzhou Gaohong Likang Biotechnology Co Ltd
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Suzhou Gaohong Likang Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The present invention provides a kind of expression respiratory syncystial virus F protein and preparation methods, wherein the amino acid sequence of the F protein is SEQ:ID:NO:1.And amino acid point mutants are carried out on the Furin recognition site of soluble F protein；GCN4 segment preparation F protein is added in C-terminal simultaneously, sequence design in this way, it ensure that the F protein expressed in eukaryotic system avoids the integrality for being digested to maintain extracellular fragment by Furin, maintain the Pre-fusion state and complete immunogenicity of F protein.Such F protein is necessary to inducing body to generate the antibody for having immanoprotection action for RSV virus, to effectively increase the probability that the later period screens positive antibody.

Description

A kind of expression respiratory syncystial virus F protein and preparation method thereof
Technical field
The present invention relates to a kind of expression respiratory syncystial virus F proteins and preparation method thereof, belong to biological medicine technology neck Domain.
Background technique
Respiratory Syncytial Virus(RSV) (Respiratory syncytial virus, RSV) belongs to Paramyxoviridae Pneumovirinae Belong to, is a kind of tunicary sub-thread minus-stranded rna virus.RSV virus will cause the whole world infection of about 64,000,000 people every year, be more than The treatment of 3000000 infants in hospital, the death of 160,000 infants are cause infant's viral bronchitis and pneumonia main Pathogen, thus be and its important for the epidemic prevention and clinical treatment of rsv infection.There is no in global range at present can The RSV vaccine of application, development is global problem.Therefore RSV virus caused by lower respiratory illness become China or even One of the disease of most serious is lacked in global infant's planned immunization.Clinically the drug for treating RSV related disease respectively has Defect, and do not listed in China, thus effectively newborn's lower respiratory illness caused by treatment RSV virus is that we urgently solve Certainly the problem of, and the therapeutic antibodies for RSV virus are to solve the problems, such as most direct means.First in antibody preparation process Step is that animal is immunized with the antigen with immunogenicity.The F protein of RSV virus is for animal immune and to induce body Generate the ideal antigen of RSV immunoprotection.F protein is a kind of most important glycoprotein of RSV virus surface, it and host cell membrane The specific receptors on surface combine, and mediate retroviral coating is merged with host cell plasma membrane, and viral nucleic acid is caused to enter host cell It is interior and replicated.Tool has the capability that mainly there are two multiple furin (Furin) since F protein is gathered around Restriction enzyme site.During application eukaryotic system expression F protein, natural activity state when its pre-fusion is kept, It is not digested by Furin, is the key that make it have intact immune originality and for animal immune.
Summary of the invention
The purpose of the present invention is to solve the above problem, proposes a kind of expression respiratory syncystial virus F protein and its preparation Method.
The purpose of the present invention is achieved through the following technical solutions:
A kind of expression respiratory syncystial virus F protein, amino acid sequence SEQ:ID:NO:1.
Preferably, a kind of preparation method for expressing respiratory syncystial virus F protein, includes the following steps:
S1, the extracellular fragment for choosing RSV virus F protein, the sequence of the extracellular fragment are SEQ:ID:NO:2, and (26AA-513AA) will Furin cutting region therein carries out point mutation, and N-terminal adds CD5 signal peptide, C-terminal adds GCN4 segment, according to the mankind's Codon preference carries out codon optimization, synthesizes the gene of the optimization, and be subcloned to pcDNA3.1-Neoless to express and carry In body, after sequence verification is errorless, endotoxin plasmid is removed using a large amount of extraction agent box preparations of Qiagen plasmid；
S2, it is transferred in centrifuge tube after choosing the defrosting of 293F cell, and the culture of FreeStyle 293 for being preheated to room temperature is added Base, room temperature is centrifuged 5 minutes after mixing, and after removing supernatant, after cell is resuspended with 293 culture medium of FreeStyle, cell is hanged Liquid is transferred in shaking flask, continuous to cultivate, adjustment cell to logarithmic growth phase；
S3, transfection reagent and the soluble F protein expression vector of above-mentioned building are uniformly mixed；Take 1 × PBS buffer solution to 6 holes It in plate, is mixed well after pcDNA-CMV-Soluble F protein plasmid is added, transfection reagent is added, mixed, it is quiet at room temperature It sets 10 minutes；
S4, it above-mentioned transfection composite is added in 293F cell mixes well, cell is placed in 37 DEG C, 5% CO2Incubator, After culture 6 ~ 8 hours, fresh 293 culture medium of FreeStyle of 50mL is added, cell is placed back in incubator and is continued Culture；
S5, after continuous culture 7 days, culture medium supernatant is collected by centrifugation, with membrane filtration, filtrate is gone in sterile centrifugation tube, is used After ion vitro immunization chromatography detection reagent detection card is detected, continue to be recombinated with palivizumab magnetic bead affinitive layer purification purpose Albumen.
Preferably, the sequence of CD5 signal peptide is SEQ:ID:NO:3 in the S1.
Preferably, the sequence of GCN4 segment is SEQ:ID:NO:4 in the S1.
Preferably, during cell suspension described in the S2 is continuously cultivated in shaking flask, when cell density reaches 2*10^6 When cell/mL, it is diluted passage, the inoculum density of cell is 5*10^5 cell/mL.
Preferably, transfection reagent is LVTransm transfection reagent in the S3.
Preferably, transfection composite is DNA/ LVTransm in the S4.
Preferably, ion vitro immunization chromatography detection reagent detection card is that Alere BinaxNOW RSV is detected in the S5 Card.
The beneficial effects are mainly reflected as follows: the F protein epitope neutralization activity due to identifying Pre-fusion state It is stronger, the structural instability of F protein, and intermediate there are the segment of Furin enzyme identification, when eukaryotic expression can be cut to F1 and Two segments of F2, in order to solve these problems, applicant carry out amino acid point on the Furin recognition site of soluble F protein Mutation；GCN4 segment is added in C-terminal simultaneously.Sequence design in this way ensure that the F expressed in eukaryotic system Albumen avoids being digested to maintain the integrality of extracellular fragment by Furin, maintains the Pre-fusion state of F protein and complete Immunogenicity.Such F protein is that body generation is induced to have the antibody institute of immanoprotection action required for RSV virus , effectively increase the probability that the later period screens positive antibody.
Detailed description of the invention
Technical scheme of the present invention is further explained with reference to the accompanying drawing:
The soluble F protein overall length schematic diagram of Fig. 1: RSV virus.
Fig. 2: SDS-PAGE electrophoresis detection result schematic diagram.
Fig. 3: Alere BinaxNOW RSV Card kit test result schematic diagram.
Fig. 4: pDonor-CMV-puro carrier schematic diagram.
Specific embodiment
Present invention discloses a kind of expression respiratory syncystial virus F proteins and preparation method thereof, wherein the ammonia of the F protein Base acid sequence is SEQ:ID:NO:1, sequence are as follows:
MELPILNTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTD AKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARREAPRGMRYTMNLQRNVNVTDSLKRKRRFLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKN NRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP LYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNL CNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYY VNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVII VILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN
Preparation method includes the following steps: in the full length sequence of RSV virus F protein, chooses the extracellular of RSV virus F protein Section (26AA-513AA), the sequence of the extracellular fragment are SEQ:ID:NO:2, sequence are as follows:
QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQ STPAANSKAKKEAPRGMRYTMNLQRNVNVTDSLKKKKKFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYML TNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEG SNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVF PSDEFDASISQVNEKINQSLAFIRKSDELL
Furin cutting region therein is subjected to point mutation, and adds CD5 signal peptide, C-terminal addition GCN4 segment in N-terminal, according to The codon preference of the mankind carries out codon optimization, synthesizes the gene of the optimization, and be subcloned to pcDNA3.1-Neoless In expression vector.Carrier removes endotoxin plasmid after sequence verification is errorless, using the big pumping kit preparation of Qiagen plasmid.Its In, the CD5 signal peptide is SEQ:ID:NO:3, the sequence are as follows: MPMGSLQPLATLYLLGMLVASCLG.The GCN4 segment Sequence is SEQ:ID:NO:4, sequence RMKQIEDKIEEILSKIYHIENEIARIKKLIGER.The mutational site is with amino Acid is seen position in the sequence of overall length, wherein the arginine R in 106 sites of coding albumen has sported coding lysine K；The arginine R for encoding 108 sites of albumen has sported coding lysine K；Encode the arginine R in 109 sites of albumen Coding lysine K is sported；The arginine R for encoding 133 sites of albumen has sported coding lysine K；Encode egg The arginine R in 135 white sites has sported coding lysine K；The arginine R for encoding 136 sites of albumen is mutated To encode lysine K.That is 106R > K, 108R > K, 109R > K, 133R > K, 135R > K, 136R > K.
It is removed from liquid nitrogen 293F cell, is thawed rapidly in 37 DEG C of water-baths, it will be after defrosting in Biohazard Safety Equipment Cell is transferred in 15mL centrifuge tube, and 293 culture medium of FreeStyle that 5mL is preheated to room temperature is added, after mixing gently, Room temperature 500xg is centrifuged 5 minutes, after removing supernatant in Biohazard Safety Equipment, is resuspended with 293 culture medium of 20mL FreeStyle After cell, cell suspension is transferred in the shaking flask of 125mL, shaking speed 130RPM, 5% CO are set2, continuous to cultivate, adjustment Cell is to logarithmic growth phase.Period when cell density reaches 2*10^6 cell/mL, is diluted passage, the inoculum density of cell For 5*10^5 cell/mL.
The soluble F protein expression vector of LVTransm transfection reagent and above-mentioned building, thaw at RT are taken out from refrigerator Afterwards, it is blown and beaten with liquid-transfering gun and is mixed completely up and down.1 × PBS buffer solution is taken out, is warmed to room temperature.Take 1 × PBS of 2mL to 6 orifice plates One hole is blown and beaten after 100 μ g pcDNA-CMV-Soluble F protein plasmids are added, above and below pipettor after mixing well, 300 μ L LVTransm are added, blows and beats mixing up and down with pipettor immediately, stands 10 minutes at room temperature.
Above-mentioned DNA/ LVTransm transfection composite is added in 50 mL 293F cells, shakes gently and mixes well. Cell is placed in 37 DEG C, 5% CO2After 130RPM is cultivated 6 ~ 8 hours, the fresh FreeStyle 293 of 50mL is added in incubator Cell is placed back in and continues to cultivate in incubator by culture medium.
After continuous culture 7 days, culture medium supernatant is collected by centrifugation, with 0.45 μm of membrane filtration, filtrate goes to sterile centrifugation Guan Zhong after being detected using Alere BinaxNOW RSV Card, is continued pure with Palivizumab magnetic bead affinity chromatography Change purpose recombinant protein.Wherein, Alere BinaxNOW RSV kit carries out the detection of soluble F protein, as a result as schemed Shown in 3, wherein SF is the detection of RSVF albumen, and Control is the detection of 293 culture medium of FreeStyle, and P is positive QC Sample detection, N are feminine gender QC sample detection, and in the above figure as the result is shown: this kit uses colloidal gold principle to be detected, There are 2 lines on nitrocellulose filter, one is sample line, is adsorbed with anti-rsv antibodies, and another is control line, is adsorbed with pair According to antibody.Anti-rsv antibodies and control antibodies are integrated on visual particle, and this particle drying is integrated to inert fiber support On.Bonding pad of the detector bar by display testing result is formed with the film by band.When test sample is added drop-wise to detector bar top In the white pad in portion, occlusion detection card.RSV antigen present in sample forms compound in conjunction with anti-rsv antibodies, this compound The anti-rsv antibodies being fixed on sample wire capture and generate color reaction, form visual sample line.Fixed control line is anti- Body captures a visual conjugate, forms a pink control line.Shown in Fig. 3, positive QC sample (sample representated by P This line and control line develop the color) and N representated by negative QC sample (control line colour developing, sample line do not develop the color) testing result show The detection of kit is in normal condition, and wherein Control is the testing result (sample of 293 culture medium of FreeStyle Line does not develop the color, control line colour developing) illustrate the substance that the testing result that has an impact is free of in culture medium, the RSVF Protein Detection result of SF (sample line and control line develop the color) shows RSV antigen positive.
SDS-PAGE detection is carried out to soluble F protein made above, testing result is as shown in Fig. 2, SDS-PAGE For detecting the size and concentration of albumen, the higher display of concentration is more obvious, it can be seen that soluble protein F's is big shown in Fig. 2 It is small in the same size in 60KD or so, with design, illustrate that the molecular weight of the soluble F protein given expression to is correct, wherein Concentration of specimens can be seen that by the amount of 2 μ g and 4 μ g as what the increase of concentration was shown is more obvious.
The application of lower F protein of the present invention described briefly below:
If it is desired to obtaining the specific nano antibody for being directed to F protein, immune alpaca is needed, but since soluble F protein has GCN4 segment needs to construct an overexpression overall length RSV F to exclude possibility of the clone of screening in conjunction with the GCN4 segment The recombination K562 cell strain of albumen；According to the overall length F protein sequence information announced on NCBI, according to the codon preference of the mankind It optimizes, subclone is into Lentiviral after synthesizing the full length fragment.In conclusion by constructing overall length F egg White wild-type sequence (full-length) and saltant type (solubility) (Furin cleavage site mutation) carry out gene chemical synthesis, sub- It is cloned into pDonor-CMV-puro, construction of expression vector.The pDonor-CMV-puro carrier is as shown in Figure 1.Carrier warp After sequence verification is errorless, endotoxin plasmid is removed using the big pumping kit preparation of Qiagen plasmid.The carrier is sleeping In beauty Transposon System carry target gene transposon vector, using the carrier and expression transposase pSB11 plasmid, Target gene stable integration can be got to the recombinant cell strain for stablizing high expression target gene into target cell genome.
Still there are many specific embodiment, all skills formed using equivalent replacement or equivalent transformation by the present invention Art scheme, all falls within the scope of protection of present invention.
Sequence table
<110>Suzhou Gao Hong Li Kang Biotechnology Co., Ltd
<120>a kind of expression respiratory syncystial virus F protein and preparation method thereof
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Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr
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Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro
225 230 235 240
Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val
245 250 255
Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu
260 265 270
Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys
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Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly
290 295 300
Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn
305 310 315 320
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325 330 335
Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser
340 345 350
Glu Val Asn Leu Cys Asn Ile Asp Ile Phe Asn Pro Lys Tyr Asp Cys
355 360 365
Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser
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Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser
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Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr
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Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr
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Arg

Claims (8)

1. a kind of expression respiratory syncystial virus F protein, it is characterised in that: its amino acid sequence is SEQ:ID:NO:1.
2. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 1, it is characterised in that: packet Include following steps:
The sequence of S1, the extracellular fragment for choosing RSV virus F protein, the extracellular fragment are SEQ:ID:NO:2, and Furin therein is cut It cuts region and carries out point mutation, and N-terminal adds CD5 signal peptide, C-terminal adds GCN4 segment, according to the codon preference of the mankind Codon optimization is carried out, synthesizes the gene of the optimization, and be subcloned the sequence verification into pcDNA3.1-Neoless expression vector After errorless, endotoxin plasmid is removed using a large amount of extraction agent box preparations of Qiagen plasmid；
S2, it is transferred in centrifuge tube after choosing the defrosting of 293F cell, and the culture of FreeStyle 293 for being preheated to room temperature is added Base, room temperature is centrifuged 5 minutes after mixing, and after removing supernatant, after cell is resuspended with 293 culture medium of FreeStyle, cell is hanged Liquid is transferred in shaking flask, continuous to cultivate, adjustment cell to logarithmic growth phase；
S3, transfection reagent and the soluble F protein expression vector of above-mentioned building are uniformly mixed；Take 1 × PBS buffer solution to 6 holes It in plate, is mixed well after pcDNA-CMV-Soluble F protein plasmid is added, transfection reagent is added, mixed, it is quiet at room temperature It sets 10 minutes；
S4, it above-mentioned transfection composite is added in 293F cell mixes well, cell is placed in 37 DEG C, 5% CO2Incubator, training After supporting 6 ~ 8 hours, fresh 293 culture medium of FreeStyle is added, cell is placed back in and continues to cultivate in incubator；
S5, after continuous culture 7 days, culture medium supernatant is collected by centrifugation, with membrane filtration, filtrate is gone in sterile centrifugation tube, is used After ion vitro immunization chromatography detection reagent detection card is detected, continue to be recombinated with palivizumab magnetic bead affinitive layer purification purpose Albumen.
3. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 2, it is characterised in that: institute The sequence for stating CD5 signal peptide in S1 is SEQ:ID:NO:3.
4. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 2, it is characterised in that: institute The sequence for stating GCN4 segment in S1 is SEQ:ID:NO:4.
5. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 2, it is characterised in that: institute State cell suspension described in S2 in shaking flask continuously cultivate during, when cell density reaches 2*10^6 cell/mL, be diluted Passage, the inoculum density of cell are 5*10^5 cell/mL.
6. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 2, it is characterised in that: institute Stating transfection reagent in S3 is LVTransm transfection reagent.
7. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 6, it is characterised in that: institute Stating transfection composite in S4 is DNA/ LVTransm.
8. a kind of preparation method for expressing respiratory syncystial virus F protein according to claim 2, it is characterised in that: institute Stating ion vitro immunization chromatography detection reagent detection card in S5 is Alere BinaxNOW RSV detection card.
CN201910095604.1A 2019-01-31 2019-01-31 A kind of expression respiratory syncystial virus F protein and preparation method thereof Pending CN109694400A (en)

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